Project 1: Over the past years, considerable excitement has accompanied the clinical testing of new drugs in melanoma. In particular, the RAF-inhibitor, PLX4032, has showed remarkable efficacy when administered to melanoma patients harboring the BRAFV600E mutation. Unfortunately, the overall clinical benefit of targeted agents in melanoma remains limited, mainly due to the appearance of resistance after a few months of treatment. Work in our lab has discovered different resistance mechanisms involving the appearance of MEK mutant alleles upon treatment of BRAFV600E cells with the MEK inhibitor CI-1040, and the overexpression of CRAF or of MAPK38/COT kinase upon treatment of BRAFV600E cells with the BRAF inhibitor PLX-4720. How general, however, these recently-discovered resistance mechanisms are, remains poorly defined, and, undoubtedly, additional mechanisms of resistance to RAF inhibition remain to be discovered. The purpose of our proposal is to perform a systematic search for novel resistance mechanisms present in, and vulnerabilities of, tumor-derived MAPK-inhibition resistant tumors. This will be addressed by 1) interrogating genetic, as well as non-genetic alterations present in patient-derived MAPK-inhibition resistant tumors, by whole-exome and transcriptome sequencing, and 2) identifying novel dependencies unique to MAPK-inhibition resistant tumors, by pooled RNAi screening, and synthetic lethal RNAi screening in the presence of MAPK-inhibitors. In addition, we will establish pre-clinical in vivo mouse models to characterize the recently discovered resistance mechanisms. Upon completion of this research, we will have gained understanding of the resistance mechanisms operant in melanoma, and identified possible new drug targets for combinatorial treatments that could overpower the resistance.